Institut de Biotechnologie des Plantes, Centre National de Recherche Scientifique Unité Mixte de Recherche 8618, Université Paris-Sud 11, 91405 Orsay Cedex, France.
Proc Natl Acad Sci U S A. 2010 Jan 5;107(1):502-7. doi: 10.1073/pnas.0910097107. Epub 2009 Dec 14.
The PII protein is a signal integrator involved in the regulation of nitrogen metabolism in bacteria and plants. Upon sensing of cellular carbon and energy availability, PII conveys the signal by interacting with target proteins, thereby modulating their biological activity. Plant PII is located to plastids; therefore, to identify new PII target proteins, PII-affinity chromatography of soluble extracts from Arabidopsis leaf chloroplasts was performed. Several proteins were retained only when Mg-ATP was present in the binding medium and they were specifically released from the resin by application of a 2-oxoglutarate-containing elution buffer. Mass spectroscopy of SDS/PAGE-resolved protein bands identified the biotin carboxyl carrier protein subunits of the plastidial acetyl-CoA carboxylase (ACCase) and three other proteins containing a similar biotin/lipoyl-binding motif as putative PII targets. ACCase is a key enzyme initiating the synthesis of fatty acids in plastids. In in vitro reconstituted assays supplemented with exogenous ATP, recombinant Arabidopsis PII inhibited chloroplastic ACCase activity, and this was completely reversed in the presence of 2-oxoglutarate, pyruvate, or oxaloacetate. The inhibitory effect was PII-dose-dependent and appeared to be PII-specific because ACCase activity was not altered in the presence of other tested proteins. PII decreased the V(max) of the ACCase reaction without altering the K(m) for acetyl-CoA. These data show that PII function has evolved between bacterial and plant systems to control the carbon metabolism pathway of fatty acid synthesis in plastids.
PII 蛋白是一种信号整合因子，参与细菌和植物氮代谢的调节。在感知细胞的碳和能量可用性后，PII 通过与靶蛋白相互作用来传递信号，从而调节它们的生物活性。植物 PII 位于质体中；因此，为了鉴定新的 PII 靶蛋白，对拟南芥叶片叶绿体可溶性提取物进行了 PII 亲和层析。只有在结合介质中存在 Mg-ATP 时，几种蛋白质才会被保留，并且它们可以通过应用含有 2-酮戊二酸的洗脱缓冲液从树脂上特异性释放。SDS/PAGE 分离的蛋白条带的质谱鉴定了质体乙酰辅酶 A 羧化酶（ACCase）的生物素羧基载体蛋白亚基和其他三个含有类似生物素/脂酰结合模体的蛋白作为潜在的 PII 靶标。ACCase 是在质体中起始脂肪酸合成的关键酶。在补充外源性 ATP 的体外重组测定中，重组拟南芥 PII 抑制质体 ACCase 活性，而在存在 2-酮戊二酸、丙酮酸或草酰乙酸的情况下，这种抑制作用完全逆转。抑制作用是 PII 剂量依赖性的，并且似乎是 PII 特异性的，因为在存在其他测试的蛋白的情况下，ACCase 活性没有改变。PII 降低了 ACCase 反应的 V(max)，而不改变乙酰辅酶 A 的 K(m)。这些数据表明，PII 功能在细菌和植物系统之间进化，以控制质体中脂肪酸合成的碳代谢途径。
